Overcoming stealthing effect of PEGylation on chitosan nanoparticles: Optimization, characterization, and assessment of cytotoxicity, apoptosis, cellular internalization, and antimetastatic efficacy.

Chitosan (CH), a natural biopolymer, has been widely utilized in nanodiagnostics and targeted cancer therapies. CH-based nanoparticles (NPs) can be directed to the tumor microenvironment (TME) via passive, active, or stimuli-responsive targeting. PEGylation is a well-acclaimed strategy to enhance NPs stability, circulation time, and passive tumor accumulation; however, excessive PEGylation can hinder cellular uptake due to the stealthing effect. To overcome these limitations, we optimized a controlled PEGylation of zoledronic acid (ZOL)-loaded CH-NPs by adjusting PEG molecular weight, density, and ZOL concentration. The optimized PEG4K-ZOL5mg-CH-NPs exhibited desirable physicochemical properties (particle size 77 ± 8 nm, PDI 0.27 ± 0.07, zeta potential 31.4 ± 3.4 mV), smooth spherical morphology, long-term stability, and sustained biphasic drug release over seven days. These NPs achieved efficient cellular internalization in MCF-7, SK-BR-3, and RAW 264.7 cells, overcoming PEG's stealthing effect by balancing prolonged circulation with enhanced uptake. Additionally, they demonstrated superior cytotoxicity, a significantly lower IC50 than unPEGylated NPs and free ZOL, enhanced apoptosis, cell cycle arrest, and reduced metastatic potential in breast cancer (BC) cells and tumor-associated macrophages (TAMs). Thus, controlled PEGylation of CH-NPs offers a promising approach for developing next-generation targeted cancer therapies with enhanced efficacy and tumor selectivity.